Application of nitroarene dioxygenases in the design of novel strains that degrade chloronitrobenzenes

Abstract

SummaryWidespread application of chloronitrobenzenes as feedstocks for the production of industrial chemicals and pharmaceuticals has resulted in extensive environmental contamination with these toxic compounds, where they pose significant risks to the health of humans and wildlife. While biotreatment in general is an attractive solution for remediation, its effectiveness is limited with chloronitrobenzenes due to the small number of strains that can effectively mineralize these compounds and their ability to degrade only select isomers. To address this need, we created engineered strains with a novel degradation pathway that reduces the total number of steps required to convert chloronitrobenzenes into compounds of central metabolism. We examined the ability of 2‐nitrotoluene 2,3‐dioxygenase from Acidovorax sp. strain JS42, nitrobenzene 1,2‐dioxygenase (NBDO) from Comamonas sp. strain JS765, as well as active‐site mutants of NBDO to generate chlorocatechols from chloronitrobenzenes, and identified the most efficient enzymes. Introduction of the wild‐type NBDO and the F293Q variant into Ralstonia sp. strain JS705, a strain carrying the modified ortho pathway for chlorocatechol metabolism, resulted in bacterial strains that were able to sustainably grow on all three chloronitrobenzene isomers without addition of co‐substrates or co‐inducers. These first‐generation engineered strains demonstrate the utility of nitroarene dioxygenases in expanding the metabolic capabilities of bacteria and provide new options for improved biotreatment of chloronitrobenzene‐contaminated sites.

Patchwork Assembly of nag -Like Nitroarene Dioxygenase Genes and the 3-Chlorocatechol Degradation Cluster for Evolution of the 2-Chloronitrobenzene Catabolism Pathway in Pseudomonas stutzeri ZWLR2-1

“…This theory is supported by a recently reported artificial evolution event in which a nitroarene dioxygenase introduced into Ralstonia sp. strain JS705, with a modified ortho pathway for chlorocatechol metabolism, resulted in the ability to grow on all three chloronitrobenzene isomers (7).…”

Application of Aromatic Hydrocarbon Dioxygenases

“…JS765 [29]. This information was further used to engineer a strain with the ability to degrade chloronitrobenzenes [70]. As observed for the activity with nitrobenzene and nitrotoluenes, substitutions at positions 258, 293, and 350 altered the activity and regi ospecificity of nitrobenzene dioxygenase toward chloronitrobenzene isomers [70].…”

“…This information was further used to engineer a strain with the ability to degrade chloronitrobenzenes [70]. As observed for the activity with nitrobenzene and nitrotoluenes, substitutions at positions 258, 293, and 350 altered the activity and regi ospecificity of nitrobenzene dioxygenase toward chloronitrobenzene isomers [70]. Substitutions at positions 258 and 350 increased oxidation at the chloro-substituted carbon, while changes at position 293 led to increased specificity with 2-and 4-chloronitrobenzene, in which nitrobenzyl alcohol production was eliminated and only chlorocatechols were generated [70].…”

“…Introduction of wild-type nitrobenzene dioxygenase and its F293Q variant led to the generation of variants of Ralstonia sp. JS705 that were able to grow on and degrade all three chloronitrobenzene isomers [70]. As opposed to known chloronitrobenzene degradation pathways that required multi ple steps for aromatic ring cleavage [75][76][77][78], the constructed pathway involved a single enzymatic step to convert chloronitrobenzene to chlorocatechols ( [70]; Figure 17.4).…”

“…JS705 that were able to grow on and degrade all three chloronitrobenzene isomers [70]. As opposed to known chloronitrobenzene degradation pathways that required multi ple steps for aromatic ring cleavage [75][76][77][78], the constructed pathway involved a single enzymatic step to convert chloronitrobenzene to chlorocatechols ( [70]; Figure 17.4). A similar naturally occurring pathway was subsequently identified in a Pseudomonas stutzeri isolate capable of growing on 2-chloronitrobenzene [79].…”